purpose. To examine the potential harmful effects on corneal structure,
innervation, and sensitivity of a spray containing the neurotoxin
capsaicin (oleoresin capsicum, OC).

methods. Ten police officers who volunteered for the study were exposed to OC.
Clinical signs were assessed. Corneal sensitivity was measured using a
Cochet–Bonnet or a noncontact esthesiometer that provides separate
measurements of mechanical, chemical, and thermal sensitivity. Tear
fluid nerve growth factor (NGF) was measured. Corneal cell layers and
subbasal nerves were examined by in vivo confocal microscopy. The
subjects were examined before application and 30 minutes, 1 day, 1
week, and 1 month after OC exposure.

results. OC spray produced occasional areas of focal epithelial cell damage that
healed within 1 day. Each eye showed conjunctival hyperemia and in two
subjects, mild chemosis. All except one eye had unchanged best
corrected visual acuity (BCVA). A transient decrease (day 1) of
mechanical sensitivity was observed with the Cochet–Bonnet
esthesiometer. With the gas esthesiometer, mechanical sensitivity
remained below normal values for 7 days. Chemical sensitivity to
CO2 was high for as much as 1 day and decreased below
normal 1 week later, whereas sensitivity to cold was unaffected. Two
subjects had measurable tear NGF that increased after exposure. Basal
epithelial cell morphology suggested temporary corneal epithelial
swelling, whereas keratocytes, endothelial cells, and subbasal nerves
remained unchanged.

conclusions. Although OC causes immediate changes in mechanical and chemical
sensitivity that may persist for a week, a single exposure to OC
appears harmless to corneal tissues. The changes are possibly
associated with damage of corneal nerve terminals of mainly
unmyelinated polymodal nociceptor fibers.

The cornea receives sensory innervation from primary sensory
neurons located in the trigeminal ganglion12345 and to a
much lesser extent from autonomic sympathetic and parasympathetic
nerves that innervate the limbus and the peripheral
cornea.6789 Corneal nerve fibers exhibit immunoreactivity
for a great variety of neuropeptides, including substance P and
calcitonin gene–related peptide (CGRP),10111213141516171819 as well as
classic neurotransmitters, presumably associated with autonomic nerve
fibers.9202122 Functionally, corneal nerve fibers have
been classified as mechanosensory units, activated only by mechanical
stimulation, polymodal units, responding also to chemical substance and
to heat, and cold-sensitive units, that are excited by low temperatures
(see Reference 23 for review).

Capsaicin (8-methyl-vanilyl-6-nonenamide), the pungent component of
chili peppers, has been shown to induce intense pain in humans and
pseudoaffective pain reactions in animals when applied to the skin and
the front of the eye, as well as neurogenic inflammation due to the
release of neuropeptides contained in nerve
terminals.242526 Capsaicin’s effects are associated with
acute stimulation of primary sensory nerve endings,2728 which is accompanied by a depletion of their neuropeptide
content.2930 This process is followed by nerve
inactivation and suppression of neurogenic inflammation in response to
injury.293132 Capsaicin also has long-lasting effects on
sensory nerves and their target tissues. Neonatal injection of
capsaicin into small rodents induces a selective sensory denervation
followed by a slow and incomplete regeneration of the sensory fibers
that is not fully compensated by increased sprouting of intact nerve
fibers and persists in adult animals.3334353637 Corneal and/or
skin ulcers and scars are concomitantly formed and may persist for
months,2934353738 resembling the clinical picture of
neuroparalytic–neurotrophic keratitis secondary to trigeminal
denervation.39 Capsaicin treatment in adult animals causes
less prominent but still detectable alterations of sensory innervation.
In vitro and in vivo studies show that nerve growth factor (NGF)
reverses the decrease of transmitter content and restores the
peripheral function of primary afferent neurons impaired by capsaicin
treatment.4041 In humans, the effects of capsaicin on
sensory innervation are poorly known. Capsaicin injected subcutaneously
produces acute pain and hyperalgesia.42 Topically applied
capsaicin ointment (0.075%) used for pain relief results in a reduced
sensitivity to cutaneous stimuli and decreased numbers of epidermal
nerve fibers. Reinnervation of the skin and recovery of sensitivity
have been observed after discontinuation of treatment.26

In the present work, we studied in human eyes the effects of topical
capsaicin on corneal innervation and corneal sensitivity using in vivo
confocal microscopy4344 and noncontact
esthesiometry.45 We also measured tear fluid NGF
concentrations in subjects exposed to capsaicin.46

Methods

OC Exposure

Testing the safety of oleoresin capsicum (OC) spray on police
officers was proposed by the Police Department of The Ministry of
Internal Affairs, Finland, with the purpose of developing
less-than-lethal or nonlethal weapons for use when arresting
individuals who resist capture. At a distance of 1.5 to 2.5 m, a
mixture containing 5.5% OC, isobutane (30.5%) as a propellant, and
isopropyl alcohol (64%) as a carrier (pepper spray, Capstum; Zarc,
Bethesda, MD) was sprayed in the face of 10 police officers who
volunteered to serve in the study. The exposure lasted for 0.5 to 1.5
seconds during which the police officers were told to hold their eyes
open. No special instructions were given in regard to breathing, but
most officers held their breath when their faces were exposed. After
the OC exposure, routine first aid was given, in which the face was
washed with cold water and soap for 5 to 15 minutes. This first aid
reduced the intensity of the pain. A medical doctor was available
during the exposure and first aid periods.

Subjects and Tests

The Police Department of the Ministry of Internal Affairs and the
Ethical Review Committee of Helsinki University Eye Hospital approved
the research plan, which followed the tenets of the Declaration of
Helsinki. Each subject gave informed consent.

The study was performed in two stages. For the first, five police
officers were used (men, 27–34 years of age). All had been exposed
several times to OC or to diorthochlorobenzyldenemalononitrile (CS):
three officers 3 times, one 5 times, and one 15 times. For the test,
subjects were treated with the OC spray and after first aid, were
queried about symptoms and examined by slit lamp biomicroscopy, corneal
photography, corneal sensitivity testing with a Cochet–Bonnet
esthesiometer, determination of tear fluid NGF levels, and in vivo
confocal microscopy. The group was re-examined 1 day, 1 week, and 1
month after OC exposure. Visual acuity was also measured at these
times. In this group of subjects, prominent changes in corneal
sensitivity were noticed. Therefore, in a second group of five police
officers (four men and one woman, 24–50 years of age), the eyes were
evaluated before and after OC exposure by the same clinical
explorations made in the previous group. Corneal sensitivity was,
however, more extensively explored using a noncontact gas
esthesiometer. Subjects were examined within the first hour, 1 day, and
1 week after OC exposure. Three subjects of this group had never been
exposed before to OC or CS. One had undergone bilateral photorefractive
keratectomy 2 years earlier. The remaining two officers had experienced
two and three previous OC exposures, respectively.

Symptom Query and Scoring of Ocular Lesions

The severity of the symptoms including nasal congestion, dyspnea,
facial hyperemia, and ocular and facial stinging pain were evaluated.
Heart rate was measured with a pulse meter (Polar Electro, Oulunsalo,
Finland) before and 1 and 10 minutes after exposure. Ocular lesions
were scored according to Draize’s scale.47

Testing of Corneal Sensitivity

Contact Sensitivity.

In the first group of subjects, the sensitivity of the central cornea
and of four corneal quadrants was explored in both eyes, by using a
Cochet–Bonnet esthesiometer provided with a number 8
filament.48 Each area was touched three times, beginning
at a filament length of 60 mm and reducing it sequentially in 5-mm
steps. A minimum of two positive answers were required for the response
to be considered positive. The longest filament that evoked the
positive response was considered as the threshold for mechanical
sensitivity.

Noncontact Gas Esthesiometry.

Corneal sensitivity of the second group of police officers was tested
unilaterally with a gas esthesiometer that performed selective
mechanical, chemical, and thermal stimulation of the
cornea.45 Gas jets of 3 seconds’ duration were applied to
the corneal surface at 2-minute intervals. Mechanical stimulation
consisted of a series of pulses of warmed air at flow rates varying
from 0 to 300 ml/min. Chemical stimulation was performed with series of
six pulses of a warmed mixture of air and CO2 at
different concentrations (0–80%). For selective thermal stimulation
10 pulses of air, warmed or cooled in the tip of the probe (from−
10o to +80°C, corresponding to changes in
corneal surface temperature between −5°C and +3°C around the
control value of 34.5°C) were used. At least one blank stimulus (a
pulse with no gas flow) was applied between pulses of each series. To
prevent mechanical stimulation during selective chemical and thermal
stimulation, flows below mechanical threshold value previously measured
for each subject were used. To avoid changes in basal corneal
temperature during selective mechanical and chemical stimulation, the
gas stream was heated up to 50°C at the tip of the
probe.45

Subjects were seated comfortably in front of a slit lamp, with the head
supported by the head holder. With the slit lamp table controls, the
tip of the gas esthesiometer was adjusted at a distance of 5 mm
perpendicular to the center of the cornea. The subject was asked to
blink immediately before the stimulus. A click produced by the opening
of the valve in the probe identified the onset of the stimulus.
Selective mechanical, chemical, and thermal stimulation was performed
in the left eye in each session. The protocol was completed after the
third session, performed 1 week after OC exposure.

Immediately after each stimulation pulse, the subject had to judge and
mark the magnitude of the various parameters of the sensation in a
continuous horizontal scale of 10 cm (the visual analog scale [VAS]).
In the VAS, 0 was assigned to no sensation and 10 to the maximal
sensation ever experienced. Six different components of the sensation
were studied: 1) intensity 2) degree of irritation, 3) magnitude of
burning pain, 4) magnitude of stinging pain, 5) cooling component of
the sensation, and 6) warming component of the sensation (Acosta MC,
Belmonte C, Gallar J, unpublished observations).

Tear Fluid Collection

Unilateral tear fluid samples were collected with a scaled 5- or
25-μl fire-polished microcapillary tube, as previously
described.49 The tear fluid flow in the collection
capillary (in microliters per minute) was calculated by dividing the
volume of the tear fluid sample by the collection time. The release (in
picograms per minute) was calculated by multiplying the concentration
in the sample by the tear fluid flow in the collection
capillary.50 As capsaicin exposure induced a marked
hypersecretion of tears, the use of the parameter “release”
(flow-corrected concentration) enabled the comparison of pre- and
postexposure concentrations. The tear samples were directly transferred
to tubes (Eppendorf, Fremont, CA) and stored at −70°C.

Tear NGF Determination

The tear NGF concentrations were measured by a two-site,
immunoenzymatic assay that recognizes human and murine NGF and is
capable of detecting as low as 5 pg/ml.46 Briefly,
polystyrene 96-well microtube immunoplates (NUNC, Napierville, IL) were
coated with affinity-purified polyclonal goat anti-NGF antibody.
Parallel wells were coated with preimmune goat IgG for evaluation of
the nonspecific signal. After overnight incubation at room temperature
and 2-hour incubation with the coating buffer (0.05 M carbonate buffer[
pH 9.5] in 2% bovine serum albumin), plates were washed with 50 mM
Tris-HCl (pH 7.4), 200 mM NaCl, 0.5% gelatin, and 0.1% Triton X-100).
After extensive washing, the diluted tear and NGF standard solutions
were distributed into the wells and left at room temperature overnight.
The plates were washed and incubated with 4 mU/well
anti-β-NGF-galactosidase (Boehringer Mannheim, Mannheim, Germany) for
2 hours at 37°C and, after another washing, 100 μl of substrate
solution (4 mg chlorophenol red/ml substrate buffer; Boehringer
Mannheim) was added to each well. After an incubation of 2 hours at
37°C, optical density was measured at 575 nm using an enzyme-linked
immunosorbent assay reader (Dynatech Laboratories, Billingshurst, UK),
and the values of standards and samples were corrected by subtracting
the background value produced by nonspecific binding. Data were
expressed in picograms per milliliter, and all assays were performed in
duplicate.

In Vivo Confocal Microscopy

A tandem scanning confocal microscope (TSCM, Model 165A; Tandem
Scanning, Reston, VA) was used to examine all layers of the central
cornea. The left eye of each subject was explored. First a drop of
topical anesthetic (benoxinate hydrochloride; Oftan Obucain;
Santen, Tampere, Finland) was applied on the cornea, and a drop of
2.5% hydroxymethylcellulose gel (Goniosol; IOLAB Pharmaceuticals,
Claremont, CA) was placed on the tip of the objective lens. The setup
and operation of the confocal microscope has been described
previously.4344 Briefly, a ×24, 0.6 numeric aperture
variable working-distance objective lens was used. The field of view
with this lens is 450 × 360 μm, and the z-axis
resolution is 9 μm. Images were detected using a low-light-level
camera (VE1000; Dage, Michigan City, IN) and recorded on SVHS tape.
Video images of interest were digitized using a computer-based imaging
system with custom software (University of Texas Southwestern Medical
Center, Dallas), and printed (Stylus Color 800 printer; Seiko Epson,
Nagano, Japan).

Statistical Analyses

Data are expressed as means ± SEM or SD. Differences between
groups or subjects were examined with parametric (Student’s t-test, one-way analysis of variance [ANOVA]) or
nonparametric statistical tests (repeated measures, ANOVA on ranks,
Friedman’s test), as necessary. Pearson correlation was used to
determine the stimulus–response relationship. Statistical significance
was set at P < 0.05.

Draize’s scale for scoring of ocular lesions includes signs in
cornea, iris, and conjunctiva.47 Six corneas of four
police officers showed focal corneal epithelial cell damage at 20
minutes as shown in Figure 1 , but none of the corneas showed opacities as described in Draize’s
scale.47 The following day, the epithelial surface of all
subjects was normal again. All eyes showed conjunctival hyperemia
(score 1) at 20 minutes. The mean duration of conjunctival injection
was 9.8 hours (range, 2–24 hours). Mild chemosis (score 1) was
observed in two subjects after the exposure, but it was undetectable
on the following day.

Visual Acuity

Best corrected visual acuity (BCVA) was not tested immediately
after OC exposure, because the subjects could not keep their eyes open.
Except for those in one subject, all eyes had unchanged BCVA (≥20/20)
throughout the study. One police officer lost one line at both 1 day
and 1 week after OC.

Sensitivity Testing

Table 1 illustrates that 20 minutes after OC exposure mechanical
sensitivity explored with the Cochet–Bonnet esthesiometer in the first
group of subjects was markedly decreased in all quadrants of the
cornea. One subject had complete bilateral corneal anesthesia, while
reduced mechanical sensitivity was observed in six eyes of the
remaining four police officers. Within 1 day, normal levels were
recovered, remaining normal to Cochet–Bonnet exploration up to 1
month. The sensitivity of right and left eyes at different time points
are shown separately.

Gas Esthesiometry

Sensation Threshold.

Table 2 presents the sensation threshold for mechanical, chemical, and thermal
stimulation of the cornea before and at 30 minutes, 1 day, and 1 week
after capsaicin treatment. Thirty minutes after OC, mechanical
sensitivity was present in all studied subjects, but the average
mechanical threshold was significantly higher than control (Table 2) ,
and the frequency distribution curve of mechanical threshold values was
shifted to the right 30 minutes, 1 day, and 1 week after OC (Fig. 2A ).

Threshold for CO2 stimulation was not
significantly modified 30 minutes after OC (Table 2) . One day later,
sensitivity to CO2 pulses was present in only
three of five subjects. In these three subjects, the
CO2 threshold was significantly higher (Table 2 , Fig. 2B ) in comparison with control values. The remaining two subjects
did not respond to CO2. One of them was again
tested 1 week after exposure to capsaicin and showed a recovered
sensitivity to CO2.

Thirty minutes after exposure to OC, sensitivity to heat had
disappeared in two subjects, but threshold for hot stimulation of the
remaining three was normal (Table 2 , Fig. 2C ). Similar results were
obtained 1 day and 1 week after capsaicin (Table 2 , Fig. 2C ). Cold
sensitivity and cold threshold were unaffected by OC (Table 2 , Fig. 2D ).

Stimulus–Response Curves.

Mechanical Stimulation.

Figure 3A illustrates the stimulus–response curve of subjective intensity of
mechanical stimulation in control conditions and 30 minutes after OC.
In both cases, subjective intensity was significantly correlated with
the magnitude of the stimulus (correlation coefficients: 0.950 and
0.966; P = 0.00,008 and 0.002 for control and 30
minutes after OC exposure, respectively). However, the values of
intensity of the sensation reported for increasing stimulus forces were
lower 30 minutes after OC (Fig. 3A) . Thus, the power of the function
describing the stimulus–response relation (Steven’s power function)
was slightly smaller for OC-treated eyes (exponent: 1.20 versus 1.54 in
control; Fig. 3A , inset). One day and 1 week after capsaicin, the
intensity curve for mechanical stimulation still deviated to the right
in comparison with control conditions (Figs. 4A ,
5 A).

Chemical Stimulation.

In control conditions, values given to the intensity of the sensation
increased with the concentration of CO2 in the
applied stimulus (Fig. 3B , squares). A significant correlation was
found between subjective intensity and magnitude of the stimulus
(correlation coefficient: 0.983, P = 0.00,001). Thirty
minutes after OC exposure, subjects reported higher VAS values for all
values of CO2 concentration (Fig. 3B , circles).
This was reflected in the steeper slope of the line obtained when the
data were fitted to a straight line (Fig. 3B , inset). Twenty-four hours
after exposure, there was no response in two individuals, whereas the
intensity–response curve to chemical stimulation of the remaining
three remained shifted to the left for the highest
CO2 concentration values (Fig. 4B) and was back
to control values 1 week after exposure (Fig. 5B) .

Hot Air.

Thirty minutes after OC exposure, no response to hot stimulation was
obtained in two subjects. VAS values reported by the remaining three
subjects were similar to control values (Fig. 3C) . Twenty-four hours
after OC exposure, responsiveness to hot air was present in two of five
subjects, with the average VAS values lower than in control subjects (Fig. 4C) . One week after exposure, values of the response to hot air
was slightly lower than in control (Fig. 5C) .

Cold Air.

Intensity–response curves obtained with cold stimulation of the cornea
were not modified by OC exposure (Figs. 3D4D5D) .

Tear Fluid NGF

NGF was at measurable levels in the tear samples of 2 of 10 police
officers (Figs. 6A 6B ). Of note, these eyes had not had earlier contacts with tear
gases, nor had they undergone any corneal surgery.

In Vivo Confocal Microscopy

In the first examination at 30 to 60 minutes after OC exposure,
surface epithelial changes were observed in two eyes. These appeared as
cells detaching from the corneal surface either individually or as a
larger cluster of highly reflective epithelial cells (Fig. 7A ). The epithelial surface regained its normal appearance by the next
day (Fig. 7B) . Although basal epithelial cells were clearly visible
during all examinations, cell borders were more pronounced 30 minutes
and 1 day after OC (Figs. 7C7D) . No morphologic changes were evident
in subbasal nerves, but they were also, in some cases, better
visualized 30 minutes and 1 day after treatment (Figs. 8A 8B, 8C ). The cornea of a police officer previously exposed to OC 15
times, showed a strange spirallike subbasal plexus with interspersing,
most probably, Langerhans’ cells (Fig. 8D) . The remaining subjects had
normal parallel, running, bifurcated, and fused nerves (Fig. 8A) . Most
of the nerves contained one or several beaded nerve fiber bundles (Fig. 8A) . The interindividual variation in visualizing subbasal nerves was
high, but the images at all time points of each individual remained
similar.

The morphology of the most anterior keratocytes remained unaltered
throughout the study (Fig. 9A 9B, 9C ). As for the basal epithelial cells, keratocyte nuclei were
also occasionally better visualized at 30 minutes. No signs of
keratocyte death or activation were observed.4451 The
morphologic appearance of the mid and posterior keratocytes as well as
the transparency was also normal throughout the study (data not shown).
The endothelial cells, easily visualized in all corneas, maintained a
regular hexagonal pattern after OC application (Fig. 9D) .

Discussion

The rationale of adding capsaicin to a self-protecting spray is
that this substance induces an intensive but relatively short-lasting
pain24 leading to blepharospasm and hypersecretion of
tears. Capsaicin sprayed onto the face immobilizes the individual and
prevents attack or resistance against arrest by law enforcement
personnel. Early-generation tear gases such as chloroacetophenone (CN)
and CS frequently induced detachment of the whole corneal epithelium
necessitating medical assistance for days or weeks. Pepper spray,
containing OC as the active substance, is widely used in private
self-protection and by the police.

This study shows that the structural and functional effects of OC spray
on conjunctiva and cornea are mild and temporary. This interpretation
applies only to the preparation used in the present work; other
solvents, such as 92% trichloroethylene, may induce severe corneal
damage (Tervo et al., unpublished observations, 1999). Several
studies on the ocular effects of capsaicin in animals have been
published.27283438 However, only recently the effects
of OC sprays on the human eye were briefly reported.52 These researchers described transient and moderate conjunctival and
corneal changes with occasional punctate epithelial erosions. In the
present study, mild and short-lasting corneal and conjunctival signs
were also observed. It is possible that epithelial damage was caused by
the alcohol solvent rather than by capsaicin, because similar grayish
white epithelial changes can occasionally be seen after contact with
isopropyl alcohol-cleaned, but wet, tonometer tips. Visual acuity was
unaffected in all but one eye, in agreement with the data of Zollman et
al.52

The acute effects of capsaicin on the sensory activity of corneal nerve
afferents in animals are well documented.272853 In the
cat eye, capsaicin at high concentrations (1%, 33 mM) produced a
transient excitation of polymodal nociceptive fibers followed by
inactivation of most fibers to all subsequent stimuli; cold sensory
fibers were weakly activated, but many remained active after capsaicin,
whereas pure mechanosensory fibers were largely unaffected by this
substance. The excitatory effects of capsaicin are due to activation of
a vanilloid receptor (VR1) present in polymodal nociceptive
neurons5455 that acts as a nonselective cationic channel,
thus depolarizing sensory nerve terminals. This strong excitatory
effect on polymodal nociceptive fibers explains the intense pain
experienced immediately after capsaicin application to the eye. Toxic
effects of capsaicin are the consequence of a massive calcium entrance
that leads to cell damage and functional inactivation, making nerve
endings insensitive to further stimuli.56

Determinations of mechanical threshold with the Cochet–Bonnet
esthesiometer after capsaicin treatment showed an acute reduction of
corneal mechanical sensitivity followed by a progressive recuperation
of control values.52 Nevertheless, the Cochet–Bonnet
esthesiometer does not permit evaluation in detail of the degree of
short- and long-term functional disturbances caused by capsaicin in the
various populations of sensory fibers that sustain corneal sensitivity.
Graded measurement of the responsiveness to mechanical, chemical, and
thermal stimulation of the cornea with the gas esthesiometer indicate
that the effect of capsaicin on the different populations of corneal
nerve fibers was heterogeneous and evolved with time.

With the gas esthesiometer, the reduction of mechanical sensitivity
observed with Cochet–Bonnet stimulation was confirmed. Responses to
low and moderate mechanical stimulation were depressed to a varying
degree among individuals 30 minutes after OC and remained below control
values 1 week after OC treatment. Acute blockade of a fraction of
polymodal nociceptors, preferentially those with unmyelinated axons
that are highly sensitive to capsaicin,53 seemed to be
responsible for the immediate reduction of sensitivity to mechanical
stimulation. Residual mechanical sensitivity in the first hour after OC
application is attributable to activation of pure mechanosensory fibers
that have a higher threshold and would be much less affected by
capsaicin27 and to those polymodal units presumably
A-delta that remained functional. The gradual return of
mechanosensitivity during the ensuing hours and days may be ascribed to
the recovery of those corneal polymodal fibers that were initially
inactivated by capsaicin. The response to chemical stimulation with
CO2, which is also mediated by polymodal fibers
was enhanced immediately after OC application. Twenty-four hours later,
it remained high in three subjects but was absent in two, reappearing
in a depressed state in all subjects 7 days after treatment. These
results confirm that a variable fraction of the polymodal fibers was
acutely inactivated and that this process reached a maximum 24 hours
after OC exposure. They further indicate that the fibers that remain
functional became sensitized57 thus producing a
hyperalgesic response. This phenomenon was more prominent with
CO2 than with heat stimulation, although both
stimuli activate polymodal nociceptive fibers, probably reflecting the
fact that heat responses are mediated by VR1 (capsaicin) receptors
while additional, capsaicin-insensitive ion channels participate in the
responses to acid.58 The absence of changes in cold
sensitivity during the 24 hours after OC exposure indicates that
cold-sensory fibers were not immediately affected by capsaicin.
Nevertheless, as occurred with the other modalities of sensation, cold
sensitivity was depressed 1 week later, implying that a part of both
polymodal and cold fibers were disturbed in the long term by the
treatment.

NGF is the prototypical member of the neurotrophin family of growth
factors.59 It plays a critical role in the development of
primary sensory neurons during embryonic life,6061 including those that innervate the cornea.62 In adult
animals, NGF receptors (TrkA) remain in the subpopulation of small
nociceptive sensory ganglion neurons.63 During chemically
induced inflammation with carrageenan64 or
turpentine,65 increased tissue levels of NGF have been
measured. Tissue NGF seems to increase the sensibility of peripheral
terminals to noxious stimuli.66 In the present experiments
two of the five police officers showed detectable levels of NGF in
tears, and levels increased after OC treatment. Elevated values could
still be measured 1 week later. In spite of the limited number of data,
these results suggest that in the cornea,67 as in other
tissues, NGF is released during noxious stimulation contributing to
sensitization and hyperalgesia of inflamed ocular tissues. Moreover,
elevated NGF levels may contribute to nerve sprouting and enhanced
neuropeptide synthesis observed in the skin after injury and in the
cornea after capsaicin treatment.3637 This in turn would
facilitate healing of the injured target tissues.386869

In vivo confocal microscopy is a noninvasive method for examining
tissue responses in different corneal sublayers of the human
cornea.4344 Toxicity of various substances has been
evaluated in an animal model,51 but to our knowledge, this
is the first in vivo confocal microscopy study on potential toxic
effects of a substance on the human cornea. The results show that OC
spray causes surface epithelial damage of short duration in some
subjects. That the cell borders of the basal epithelial cells were
easily visualized at 30 minutes and 1 day after OC, without signs of
cell damage, suggests epithelial swelling.

No changes could be ascertained in the morphology of subbasal nerves
after a single pepper spray exposure. Electron microscopic
observations7071 have revealed that corneal subbasal
nerves that are seen by in vivo confocal microscopy correspond to nerve
bundles, because visualization of individual nerve fibers is beyond the
level of resolution of confocal microscopy. In most cases the nerves
were more apparent after OC exposure, probably because of swelling of
the epithelial cells through which the nerves are pressed into the same
focal plane. The images of the nerve fiber bundles did not vary during
the study, and no signs of sprouting were apparent. It is possible that
the insult to the nerves is not great enough to induce sprouting.
Alternatively, sprouting may be beyond the level of resolution or is
limited to the peripheral cornea, as described for experimental
animals,37 which was out of the range of observation in
the present experiment in which explorations were limited to the
central cornea. A surprising finding was the spirallike nerve fiber
bundle arrangement of the subbasal plexus in the eyes of a police
officer repeatedly exposed to OC or CS. A similar organization has been
observed in the nerves of the cornea of an alkaline phosphatase
transgenic mice (Belmonte and Raviola, unpublished
observations, 1999), but its significance is obscure.

Because of the mild and transient signs of tissue injury, it can be
concluded that single exposure of human eyes to OC is relatively
harmless to the cornea and conjunctiva. However, one should be cautious
in repeated OC exposures, because long-lasting changes in corneal
sensitivity could occur. These changes are possibly associated with
damage of nerve terminals of mainly unmyelinated polymodal nociceptive
fibers.

Data are Cochet–Bonnet esthesiometer filament lengths (mean ± SD). The differences in sensitivity of each area at various time
points did not reach statistical significance (Friedman’s test). Right
(OD) and left (OS) eyes considered separately. Pre-exposure sensitivity
values are not available.

Cumulative distribution of thresholds for mechanical (A),
chemical (B), hot (C), and cold (D)
selective stimulation of the cornea of human volunteers before and at
different times after OC application. Data represent the incidence of
positive responses of subjects to increasing intensities of stimulation
as a percentage of the total number of stimuli. Lines of increasing
thickness represent control, 30 minutes after OC, 1 day after OC, and 1
week after OC.

Figure 2.

Cumulative distribution of thresholds for mechanical (A),
chemical (B), hot (C), and cold (D)
selective stimulation of the cornea of human volunteers before and at
different times after OC application. Data represent the incidence of
positive responses of subjects to increasing intensities of stimulation
as a percentage of the total number of stimuli. Lines of increasing
thickness represent control, 30 minutes after OC, 1 day after OC, and 1
week after OC.

Corneal epithelial cells after OC exposure. A large cluster of highly
reflective surface epithelial cell is observed in one cornea
immediately after exposure (A). The epithelium appeared
close to normal on the following day (B). Basal epithelial
cells were clearly perceived before exposure (C), but at 30
minutes the cell borders were more pronounced, possibly because of
epithelial swelling (D). Image size, 265 × 220 μm.

Figure 7.

Corneal epithelial cells after OC exposure. A large cluster of highly
reflective surface epithelial cell is observed in one cornea
immediately after exposure (A). The epithelium appeared
close to normal on the following day (B). Basal epithelial
cells were clearly perceived before exposure (C), but at 30
minutes the cell borders were more pronounced, possibly because of
epithelial swelling (D). Image size, 265 × 220 μm.

Subbasal nerves after OC exposure. No morphologic changes could be
detected by confocal microscopy (A) before exposure,
(B) at 30 minutes, and (C) at 1 day. However, a
cornea with 15 previous exposures to OC or other tear gases showed an
abnormal spirallike subbasal plexus, not previously described in humans
(D). Image size, 380 × 275 μm.

Figure 8.

Subbasal nerves after OC exposure. No morphologic changes could be
detected by confocal microscopy (A) before exposure,
(B) at 30 minutes, and (C) at 1 day. However, a
cornea with 15 previous exposures to OC or other tear gases showed an
abnormal spirallike subbasal plexus, not previously described in humans
(D). Image size, 380 × 275 μm.

The morphology of the first anterior keratocytes after OC exposure
remained the same, and no signs of activation could be detected
(A) before exposure, (B) at 30 minutes, and
(C) at 1 day. Only the keratocyte nuclei can be perceived.
Endothelial cells showed a regular hexagonal pattern throughout the
follow-up (D, at 1 month). Image size, 265 × 220μ
m.

Figure 9.

The morphology of the first anterior keratocytes after OC exposure
remained the same, and no signs of activation could be detected
(A) before exposure, (B) at 30 minutes, and
(C) at 1 day. Only the keratocyte nuclei can be perceived.
Endothelial cells showed a regular hexagonal pattern throughout the
follow-up (D, at 1 month). Image size, 265 × 220μ
m.

Maggi CA, Santicioli P, Geppeti P, et al. Involvement of a peripheral site of action in the early phase of neuropeptide depletion following capsaicin desensitization. Brain Res. 1987;436:402–406.[CrossRef][PubMed]

Cumulative distribution of thresholds for mechanical (A),
chemical (B), hot (C), and cold (D)
selective stimulation of the cornea of human volunteers before and at
different times after OC application. Data represent the incidence of
positive responses of subjects to increasing intensities of stimulation
as a percentage of the total number of stimuli. Lines of increasing
thickness represent control, 30 minutes after OC, 1 day after OC, and 1
week after OC.

Figure 2.

Cumulative distribution of thresholds for mechanical (A),
chemical (B), hot (C), and cold (D)
selective stimulation of the cornea of human volunteers before and at
different times after OC application. Data represent the incidence of
positive responses of subjects to increasing intensities of stimulation
as a percentage of the total number of stimuli. Lines of increasing
thickness represent control, 30 minutes after OC, 1 day after OC, and 1
week after OC.

Corneal epithelial cells after OC exposure. A large cluster of highly
reflective surface epithelial cell is observed in one cornea
immediately after exposure (A). The epithelium appeared
close to normal on the following day (B). Basal epithelial
cells were clearly perceived before exposure (C), but at 30
minutes the cell borders were more pronounced, possibly because of
epithelial swelling (D). Image size, 265 × 220 μm.

Figure 7.

Corneal epithelial cells after OC exposure. A large cluster of highly
reflective surface epithelial cell is observed in one cornea
immediately after exposure (A). The epithelium appeared
close to normal on the following day (B). Basal epithelial
cells were clearly perceived before exposure (C), but at 30
minutes the cell borders were more pronounced, possibly because of
epithelial swelling (D). Image size, 265 × 220 μm.

Subbasal nerves after OC exposure. No morphologic changes could be
detected by confocal microscopy (A) before exposure,
(B) at 30 minutes, and (C) at 1 day. However, a
cornea with 15 previous exposures to OC or other tear gases showed an
abnormal spirallike subbasal plexus, not previously described in humans
(D). Image size, 380 × 275 μm.

Figure 8.

Subbasal nerves after OC exposure. No morphologic changes could be
detected by confocal microscopy (A) before exposure,
(B) at 30 minutes, and (C) at 1 day. However, a
cornea with 15 previous exposures to OC or other tear gases showed an
abnormal spirallike subbasal plexus, not previously described in humans
(D). Image size, 380 × 275 μm.

The morphology of the first anterior keratocytes after OC exposure
remained the same, and no signs of activation could be detected
(A) before exposure, (B) at 30 minutes, and
(C) at 1 day. Only the keratocyte nuclei can be perceived.
Endothelial cells showed a regular hexagonal pattern throughout the
follow-up (D, at 1 month). Image size, 265 × 220μ
m.

Figure 9.

The morphology of the first anterior keratocytes after OC exposure
remained the same, and no signs of activation could be detected
(A) before exposure, (B) at 30 minutes, and
(C) at 1 day. Only the keratocyte nuclei can be perceived.
Endothelial cells showed a regular hexagonal pattern throughout the
follow-up (D, at 1 month). Image size, 265 × 220μ
m.

Data are Cochet–Bonnet esthesiometer filament lengths (mean ± SD). The differences in sensitivity of each area at various time
points did not reach statistical significance (Friedman’s test). Right
(OD) and left (OS) eyes considered separately. Pre-exposure sensitivity
values are not available.